This application is a 371 of PCT/EP98/01789 filed on Mar. 26, 1998 which claims foreign priority to German Applications 19713093.3 filed on Mar. 27, 1997 and 19713094.1 filed Mar. 27, 1997.
The subject of this invention are homogeneous formulations containing glycerophospholipids and polar or lipophilic substances, and a method of producing a these formulations.
Glycerophospholipids play an important physiological role as building blocks for membranes, especially during compartmentation in biological systems. They are accordingly ubiquitous in animal, plant and microbial forms of life.
Chemically speaking, natural glycerophospholipids consist of glycerol which is esterified in the C1 and C2 positions with fatty acids and carries a phosphatide ester in the C3 position. From the point of view of quantity, by far the most important natural glycerophospholipids are the phosphatidyl derivatives phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol and phosphatidic acid. In certain cell systems, however, there are, in addition, high concentrations of other phospholipids, eg, plasmalogens, cardiolipins or sphingomyelins.
In principle, it is possible to synthesise glycerophospholipids in vitro by means of chemical and/or enzymatic processes. The structure of the products obtained can correspond to that of natural glycerophospholipids, but can also be xe2x80x9csyntheticxe2x80x9d. However, at the present time, biological sources still take precedence in industrial-scale production because they are readily available. This applies especially to plant lecithins, eg, from soybeans, rape or sunflower seed. These lecithins are obtained as a mixture of various phospholipid classes, so-called xe2x80x9craw lecithinxe2x80x9d, in the refining process during the production of cooking oils. The most important animal source of glycerophospholipids is the yolk from hens"" eggs, which is characterized by a high phosphatidyl choline content. There are various methods available, eg, solvent extraction, with which it is possible to increase the proportion of certain glycerophospholipid classes in the naturally occurring mixtures, and/or, usually with the help of chromatographic separation methods, to obtain them in uniform form; here too, phosphatidyl choline is particularly important.
By means of chemical and/or enzymatic processes, eg, by means of hydrolysis, hydroxylation or hydrogenation, naturally occurring glycerophospholipids can be modified such that their surface-active properties, in particular, are changed.
The main reason why surface-active glycerophospholipids are used technologically is because of their emulsifying effect, which is exploited specifically to stabilise emulsions or suspensions, eg, traditionally in the food-processing sector, in industry, and in the pharmaceuticals sector. In addition, glycerophospholipids can also be used physiologically, because in vivo they fulfil important functions, especially as building blocks for membranes in biological cells. By virtue of this fact, and because they are toxically innocuous, natural glycerophospholipids in particular, especially phosphatidyl cholines and cephalins, are used for products or formulations which can be supplied directly or indirectly to humans. Of particular interest is their use in food and in pharmaceutical products. It is also known that the resorption, pharmacokinetics and/or the pharmacological effect of active ingredients used in drugs can be varied by formulating them with glycerophospholipids.
In the preparation of emulsions and dispersions, eg, in the formulation of pharmaceutical active ingredients, but also in other technical areas, it is often desirable for the active ingredients to be dispersed as finely and homogeneously as possible. This necessitates, eg, a small particle size. Formulations and methods of producing them with which the active ingredient can be more finely distributed by way of the formulation than was possible so far are accordingly of great interest and can be of far-reaching importance.
According to the prior art, there are three main methods of formulating glycerophospholipids with polar or lipophilic substances:
(1) formulation of the (dissolved) polar substances or (liquid or dissolved) lipophilic substances in membrane-forming glycerophospholipid vesicles or liposomes (xe2x80x9cmicellar systemxe2x80x9d),
(2) formulation by means of incorporating fine (micro-crystalline) particles of the solid polar or lipophilic substance in glycerophospholipids (xe2x80x9cmicro-crystalline systemxe2x80x9d), and
(3) formulation by means of binding the polar or lipophilic substance chemically to glycerophospholipids (chemical-bond system).
In xe2x80x9cmicellar-systemxe2x80x9d formulations, the polar substances are dissolved in polar solvents, usually water, and are surrounded by a single- or multi-layer membrane, eg, a bilayer membrane, consisting of surface-active glycerophospholipids. The lipophilic substances, in liquid form or dissolved in suitable solvents, are encapusulated in a similar way. A recent survey on this formulation technique is contained in H. Hauser, Phospholipid Vesicles in Cevc. G. (ed.), Phospholipid Handbook, Marcel Dekker, New York, 1993, pp. 603-637. Building up a micellar bilayer membrane for the formulation of polar substances in water or of lipophilic substances is, however, not unproblematic, and certain esters of phosphoric acid, eg, in the form of glycerophospholipids such as phosphatidyl choline, may have to be present in the membranes in order to impart the required stability.
The inclusion rates for the active ingredients to be formulated are often low, so that large active-ingredient losses have to be allowed for during production of the formulation. In addition, sterol derivatives such as cholesterol are often needed to stabilise the membrane. Attempts have also been made to stabilise the membrane by forming an adductxe2x80x94by means of a chemical bondxe2x80x94between cholesterol and the substance to be formulated (eg, J. L. Murtha et al., J. Pharm. Sci 83 (9), 1222-8, 1994). Another approach has been to produce liposomal formulations using supercritical carbon dioxide, rendering the use of large volumes of organic solvent unnecessary (Frederiksen L. et al, J. Pharmaceutical. Sci. 86, 921-8, 1997). Formulations of active ingredients, eg, through use of the liposome technique, have resulted in great progress, eg, in many therapies used in modern medicine; however, there are two main disadvantages, eg, in parenteral applications: for one, liposomesxe2x80x94as artificial micellesxe2x80x94have only a limited lifetime in vivo because lipid exchange reactions, in particular, can take place at membranes and thus destabilise the membranous vesicle or liposome, or even cause it to disintegrate, before it reaches the actual place of intended therapy. For another, especially in the case of larger micelles, the mononuclear phagocyte system becomes active, and leads to undesired immunological side reactions. For physical reasons, it is impossible to reduce the size of vesicular liposomes arbitrarily and thus to avoid the immune response, because the surface of the membrane, depending on its composition, will tear open as from a certain micelle size.
In formulations where polar or lipophilic substances are incorporated in micro-crystalline form in glycerophospholipids, the problem likewise arises that according to prior art, the size of the microcrystals cannot be arbitrarily reduced. If a solvent system is used, for example, in which both the polar/lipophilic substances and the glycerophospholipids are dissolved, and this solution is subjected, eg, to spray drying, the solubility limits of the substances and the glycerophospholipids are reached at different solvent concentrations during removal of the solvent, and a xe2x80x9cmicrocrystalline formulationxe2x80x9d is obtained. As the time needed to remove the solvent approaches zero, the size of the particles could, in theory, be reduced still further, but this is technically unfeasible.
The patent specification EP-B-O 493 578 describes the use of supercritical carbon dioxide to try and overcome these problems. For this process, however, both the emulsifier and the substance to be formulated must be soluble in supercritical carbon dioxide so that the requisite homogeneous distribution can be obtained. But the solubility of glycerophospholipids in supercritical carbon dioxide is only marginal, and only selected active ingredients can be used in this process. The first of these two disadvantages alone is sufficient to render the proposed method of formulating active ingredients with glycerophospholipids unsuitable.
Another method of producing a powdery drug form comprising one or more active ingredients and one or more carriers, which is conducted with the help of a fluid gas, is known from the EP-A-O 322 687. According to this method, a liquid medium containing the active-ingredient component and the carrier component is first of all brought into contact with the fluid gas; the liquid is then removed by the fluid gas, and an active ingredient/carrier preparation obtained which is free of liquid medium. By means of this process, in which, eg, CO2, N2O and hydro-carbons in supercritical state can be used as fluid gases, and polymers, lipids, lecithins and waxes as carrier components, small hollow spheres are obtained in powder form by means of so-called spray-embedding. The liquid components contained in the formulation are separated completely from the preparation by means of a spray tower and one- or multi-component nozzles, and the preparation is obtained in powder form. The disadvantages of this xe2x80x9cspray-embeddingxe2x80x9d process are, on the one hand, the technical complexity:
(i) The value-determining particle size of the solid formulation obtained is determined by a complicated nozzle design which is susceptible to technical problems;
(ii) Experience has shown that it is technically very difficult to remove extremely fine powder from the spray tower, because it is hardly practicable to collect the fine particles in a separator.
On the other hand, it is a disadvantage if all the liquid components (ie, also any liquid formulation aids that may be present) have to be completely removed, because the microcrystalline powder obtained is only suitable for specific applications, eg, for the production of micro-encapsulations in polymers, with delayed drug release.
Formulations in which a substance of polar or lipophilic character is bound chemically to surface-active glycerophospholipids (xe2x80x9cchemical-bond systemxe2x80x9d), of the type described, eg, by Hong et al. in Cancer Res. 50 (14), 4401-6 (1990), have the major disadvantage that this method is complicated and not generally applicable, an added problem being the fact that on account of the chemical bond, the substance""s mode of action usually changes. Accordingly, this formulation strategy is limited in practice to just a few exceptional cases.
The general objective of an improved formulation, namely to obtain a molecularly disperse distribution between active ingredient and glycerophospholipid by means of molecular, preferably noncovalent interaction, is not achieved with the procedures described above.
Appropriate solutions to the problems outlined above are not known according to the prior art, and approaches to a solution have only been described for formulations of water-insoluble, non-polar substances (U.S. Pat. No. 4,973,465 or WO 91/16 068). It is thus of considerable interest to further improve active-ingredient distribution, especially in pharmaceutical formulations.
The object of this invention was thus to provide homogeneous, anhydrous formulations comprising active ingredients, carriers, and maybe formulation aids, which do not have the described disadvantages of hitherto known formulations and which, in particular, reduce or completely prevent the formation of micro-crystalline particles.
This objective was established by means of homogeneous, anhydrous formulations containing
(A) one or more glycerophospholipids
(B) one or more polar and/or lipophilic substances showing an affinity with glycerophospholipids
(C) maybe one or more formulation aids with at least two hydroxyl groups, characterized in that the glycerophospholipid component (A) and the component (B) are present in a molar ratio of 1:0.001 to 2 andxe2x80x94in cases where (C) is presentxe2x80x94the glycerophospholipid component (A) and the component (C) are present in a molar ratio of 1:0.001 to 1.
In this invention, glycerophospholipids are understood to be compounds containing a glycerol radical which is esterified with at least one fatty acid radical and with at least one phosphatide radical. Examples of suitable classes of glycerophospholipids include phosphatidic acids, phosphatidyl esters, lyso-phospholipids, cardiolipins and plasmalogens. Preference is given to phosphatidyl esters and lyso-phospholipids which are esterified with a fatty acid radical at the C1 atom of the glycerol, and with a phosphatide radical at the C3 atom of the glycerol. It is especially beneficial to select the glycerophospholipids (A) from compounds which have the general formula (I): 
where R1 and R2 can be the same or different, and each stand for a fatty acid radical with the general formula (II): 
where R stands for straight-chain or branched-chain, saturated or mono- or polyunsaturated C6 to C24 fatty acid radicals, which may be substituted in the chain with, eg, OH or heteroatoms such as O, N or S, where R2 can also be H, and where X is a radical from the series xe2x80x94H, CH2xe2x80x94CH2xe2x80x94NH3+, xe2x80x94CH2xe2x80x94CH2xe2x80x94Nxe2x80x94(CH3)3+, xe2x80x94CH2xe2x80x94CH(NH3+)COOxe2x88x92, xe2x80x94CH2xe2x80x94CH(xe2x80x94OH)xe2x80x94CH2xe2x80x94OH or 
The average molecular weights of the glycerophospholipids can be used as a basis for calculating the molar ratios.
Surprisingly, it was found that when introduced into hydrophilic and/or lipophilic media and into two-phase systems, the formulations of the invention show a high level of stability and either do not dissociate into the starting components, or do so only to a minor extent. The formulations of the invention show solution behaviour which can be controlled selectively by way of the molar ratio of glycerophospholipid (A) to the polar or lipophilic substance (B): with an equimolar ratio of glycerophospholipid to polar substance, one possibility provided for according to the invention, the formulations are amphiphilic, ie, in a two-phase system they have a surface-active effect. If there is an excess of glycerophospholipid, these formulations are more lipophilic, ie, they can be incorporated well in oil. Water-soluble colouring agents, for example, which have been formulated with an excess of glycerophospholipid, form clear, coloured solutions in oil at temperatures  greater than 50xc2x0 C. Through additional use of the formulation aid (C), the dispersibility and/or solubility in water, as well as the stability of the emulsion, can be decisively improved in all formulations of the invention. This is evident, for example, in formulation-aid-containing formulations of non-polar active ingredients, which, at temperatures  greater than 50xc2x0 C., can be dispersed readily in water to form a stable emulsion; the formulation can be sterilised by means of a 0.2 xcexcm filter without deaggregating. In the formulation of lipophilic substances, the solubility of the overall formulation in water is improved significantly.
Especially good properties in terms of the invention are shown by formulations which contain lecithins and/or cephalins as glycerophospholipid component (A).
As substances (B) with polar and/or lipophilic properties, it is preferable according to the invention to use physiological active ingredients or colouring agents, the molecular weight of which should be  less than 1500 daltons, and, in particular,  less than 500 daltons.
An essential aspect is that the substances (B) have an affinity with glycerophospholipids, this affinity not necessarily resulting in a covalent chemical reaction butxe2x80x94as is especially preferred in this casexe2x80x94being manifested in the formation of non-covalent interactions such as secondary valences, so-called hydrogen bonds, and/or lipophilic interactions. As this happens the system is stabilised, with formation of the homogeneous, anhydrous aggregates (xe2x80x9cmolecular self-assembliesxe2x80x9d) which are typical of the formulations of the invention and, as far as their structure is concerned, compare best with xe2x80x9csolid solutionsxe2x80x9d.
In this invention, physiological active ingredients are understood to be all compounds or classes of substances which have a regulating or controlling influence on metabolic processes. Especially when administered to mammals or humans, the effect is naturally dependent on the form in which the active ingredient is administered. The term xe2x80x9cpolar substancexe2x80x9d or xe2x80x9clipophilic substancexe2x80x9d accordingly covers all polar and also some water-soluble or lipophilic active ingredients in drugs which are suitable for topical and transdermal applications, or which can be administered through the mouth, parenterally or by way of inhalation and, in particular here, intravenously, intramuscularly, subcutaneously, intraperitoneally or intranasally.
The term also includes active ingredients used in cosmetics, and, in addition, agrochemicals such as fertilisers, plant growth regulators, herbicides and insecticides, and also biocides of general nature.
Of the large variety of physiological active ingredients in question, the water-soluble vitamins or fat-soluble vitamins, such as those of the groups A, D, E and K, are quoted by way of example. An example of the water-soluble colouring agents is Ponceau 4 R (E 124), while the carotinoids are important examples of the lipophilic colouring agents.
The formulations of the invention are anhydrous, ie, they preferably contain less than 5 wt. %, better still less than 3 wt. % and best of all less than 1 wt. % of water, expressed in terms of the overall weight of the formulation.
In addition, it is beneficial if the formulations of the invention are oil-free, preferably containing a maximum of 3 wt. % and, better still, a maximum of 2 wt. % of oil, ie, triglycerides, expressed in terms of the overall weight of the formulation. It is of particular advantage if the formulations of the invention are solid at room temperature, eg, in the form of a free-flowing powder.
According to the invention, the formulations may also contain one or more polyol compounds as formulations aids (C), the polyols pereferably being liquid polyols, especially C2-C4 compounds, and containing at least two hydroxyl groups. The greatest preference is given here to glycerol. The polyols in question can also be used in the form of arbitrary mixtures. It is of advantage, however, if the formulation aids for preparing the formulation are anhydrous, ie, preferably having a maximum water content of 5 wt. %, expressed in terms of the weight of the formulation aid.
In addition to the formulations, this invention includes a method of producing them, which is carried out in three main steps:
First of all, in step (a), substance (B) is provided in liquid form, eg, as a pure substance, assuming it is liquid under the conditions in question. It is preferable, however, if substance (B) is dissolved in an anhydrous solvent (mixture), ie, a solvent which preferably contains a maximum of 5 wt. % water. Polar and/or non-polar solvent (mixtures) are especially suitable for this purpose. It is also possible to admix the formulation aid (C), which again is anhydrous, with the solvent (mixture) in the molar ratios given. If a polar substance is to be formulated, this substance can also be dissolved exclusively in the anhydrous formulation aid.
The polar solvents provided for in the invention are solvents of protic and/or aprotic character. As protic solvents, primary monovalent C1-10 alcohols, secondary monovalent C3xe2x88x9210 alcohols and tertiary monovalent C4-10 alcohols, as well as arbitrary mixtures thereof, have proved to be especially suitable. Preferred examples of aprotic solvents are halogenated C1-10 hydrocarbons, especially chloroform, as well as ethers such as diethyl ether and tetrahydrofuran (THF), and also arbitrary mixtures thereof.
Suitable non-polar solvents are aliphatic or cyclic C5-10 hydrocarbons, and/or triglycerides.
In special cases, it is useful in step (a) to use mixtures of polar and non-polar solvent (mixtures) in a weight ratio of max. 1:1.
The liquid or solution resulting from step (a) is then combined in step (b) with a glycerophospholipid (mixture) (A) in such a way that the dissolved state of the components is maintained. The prerequisite for this is that the glycerophospholipid component (A) is also used in the dissolved state, for which purpose, once again, anhydrous polarxe2x80x94but also non-polarxe2x80x94solvents or mixtures thereof are especially suitable. In addition, a formulation aid (C) may be present.
Should it be necessary to use non-polar solvents in order to obtain a solution, aliphatic or cyclic C5-10 hydrocarbons, preferably hexane and/or cyclohexane, and/or triglycerides, have proved suitable in steps (a) and/or (b), as was mentioned before. From the group of triglycerides, preference is given in the invention especially to natural vegetable oils.
The mixture of dissolved substances resulting from step (b) is subsequently subjected to an extraction process in order to remove the solvent (mixture). For the extraction, it is of advantage to use an extracting agent containing hydrocarbons such as propane and/or butane, which are gaseous under normal conditions. According to an especially preferred embodiment, the extraction is carried out in a rectifying column under a pressure between 1 and 50 MPa and at a temperature from 20 to 150xc2x0 C., using an extracting agent containing propane and/or butane; the extraction is conducted in such a manner that the extraction mixture is distributed between a homogeneous lower phase comprising glycerophospholipid (mixture) (A), substance (B) and maybe formulation aid (C), and an upper phase containing the solvent and maybe the substance (B), and that the lower phase separates from the upper phase, with the formulation being obtained from the lower phase, which is generally in the form of a melt. The method of the invention for producing the formulation can thus be carried out with no significant loss of the substance to be formulated.
Under the conditions preferred according to the invention, the solution of starting materials obtained from steps (a) and (b) is extracted in a rectifying column by an extracting agent (mixture) coming from the bottom and preferably consisting of propane with up to 95 wt. % dimethyl ether (DME).
The extraction conditions preferred according to the invention are a pressure between 1 and 50 MPa and a temperature of 20 to 150xc2x0 C., with pressures in the range between 3 and 20 MPa and temperatures of 30 to 100xc2x0 C. having proved especially suitable. Following extraction, which is preferably conducted as a continuous, countercurrent process, the extracting agent (mixture) is conducted away, while the glycerophospholipid fraction, sinking in the form of a melt to the bottom of the column, takes up the substance (B) to be formulated, and also the formulation aid, if one was used; it is at this stage that the formulation containing the glycerophospholipid (mixture), the polar or lipophilic substance and, if one was used, the formulation aid, is actually formed, and this can take place entirely in the dissolved state. For improved separation of the starting mixture into a liquid upper phase and a lower phase (melt), it has proved highly beneficial to operate with a temperature gradient in the column, the temperature at the top being 5 to 50xc2x0 C. higher than that at the bottom.
The fused formulation at the bottom of the column, which usually contains between 20 and 40 wt. % of extracting agent (mixture), can be discharged via a suitable arrangement of nozzles into an ambient-pressure environment and thus freed of extracting agent (mixture) by means of the resulting pressure reduction and/or an increase in temperature. The solvent (mixture) obtained as top product is likewise freed of extracting agent (mixture), again by means of a pressure reduction and/or an increase in temperature. For this purpose it is expedient to use a separator.
It is also possible to compress the extracting agent (mixture) and recycle it.
During production of the formulations of the invention in the manner described, it is especially beneficial if the fused glycerophospholipid (mixture) (A) can take up the substance (B), which is to be formulated, directly from the solvent (mixture) of the feed mixture. By virtue of the procedure of the invention and in contrast to hitherto known processes, no microcrystalline particles are formed. This explains why the claimed formulations are fully homogeneous.
Very generally, therefore, the method of the invention serves to transform substances of polar and/or lipophilic character into homogeneous formulations with lipophilic or amphiphilic properties, so that they can be used for applications which up till now were very difficult or completely impracticable.
This also explains why these homogeneous formulations, in keeping with the idea behind the invention, are especially suitable for the preparation of dispersions, emulsions and/or suspensions for the food processing industry, biotechnology, the agrochemicals, cosmetics and pharmaceuticals industriesxe2x80x94here in particular for the formulation of active ingredientsxe2x80x94but also for the paint and varnish industry and the leather industry.
Additional subject matter of the invention is a pharmaceutical preparation containing a formulation according to the invention, maybe together with carriers, aids, fillers and/or diluents such as are common in the pharmaceuticals industry.
Of particular interest here is the distribution in the claimed, stable formulations or aggregates with glycerophospholipids beneath the critical micelle concentration. These new possibilities are especially important for membrane-penetration processes.